CRISPR/Cas9-Mediated genomic knock out of tyrosine hydroxylase and yellow genes in cricket Gryllus bimaculatus

Gryllus bimaculatus is an emerging model organism in various fields of biology such as behavior, neurology, physiology and genetics. Recently, application of reverse genetics provides an opportunity of understanding the functional genomics and manipulating gene regulation networks with specific physiological response in G. bimaculatus. By using CRISPR/Cas9 system in G. bimaculatus, we present an efficient knockdown of Tyrosine hydroxylase (TH) and yellow-y, which are involved in insect melanin and catecholamine-biosynthesis pathway. As an enzyme catalyzing the conversion of tyrosine to 3,4-dihydroxyphenylalanine, TH confines the first step reaction in the pathway. Yellow protein (dopachrome conversion enzyme, DCE) is also involved in the melanin biosynthetic pathway. The regulation system and molecular mechanism of melanin biogenesis in the pigmentation and their physiological functions in G. bimaculatus hasn’t been well defined by far for lacking of in vivo models. Deletion and insertion of nucleotides in target sites of both TH and Yellow are detected in both F0 individuals and the inheritable F1 progenies. We confirm that TH and yellow-y are down-regulated in mutants by quantitative real-time PCR analysis. Compared with the control group, mutations of TH and yellow-y genes result in defects in pigmentation. Most F0 nymphs with mutations of TH gene die by the first instar, and the only adult had significant defects in the wings and legs. However, we could not get any homozygotes of TH mutants for all the F2 die by the first instar. Therefore, TH gene is very important for the growth and development of G. bimaculatus. When the yellow-y gene is knocked out, 71.43% of G. bimaculatus are light brown, with a slight mosaic on the abdomen. The yellow-y gene can be inherited stably through hybridization experiment with no obvious phenotype except lighter cuticular color. The present loss of function study indicates the essential roles of TH and yellow in pigmentation, and TH possesses profound and extensive effects of dopamine synthesis in embryonic development in G. bimaculatus.


Introduction
Crickets are typical singing insects belonging to Orthoptera with over 6000 extant species. They have been used as a classical system on speciation and acoustic communication [1,2]. To

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PLOS ONE | https://doi.org/10.1371/journal.pone.0284124 April 10, 2023 1 / 17 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 study these issues, a representative model species needs to be selected. Ease of breeding [3], large number of eggs [3], detailed embryo development stage tables [4] and assembled and annotated genome [5] are the advantages of G. bimaculatus (Orthoptera: Gryllidae). In addition, it has been found that the isolated protein of G. bimaculatus can be used as an osteoinductive material for the fabrication of osteoinductive scaffolds or as an alternative to expensive protein supplements to treat bone-related diseases [6]. Thus, G. bimaculatus is an emerging model organism in various fields of biology such as behavior, neurology, physiology and genetics [3,7]. It has been becoming a highly amenable hemimetabolous insect in laboratory [8].
The reverse genetics technology is essential in the development and physiology research of G. bimaculatus. At present, there are mainly RNA interference (RNAi), TALENs and the Clustered Regularly Interspaced Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/ Cas9) gene editing technology in the research methods of gene function of G. bimaculatus. For example, RNAi was used to study tissue-specific regeneration molecular pathways such as JAK/STAT signaling pathways involved in the legs regeneration of G. bimaculatus [9]. However, RNAi efficiency can vary between different insect groups, developmental stages or tissues and due to its transient characteristic, it might not be suitable for studying some candidate genes [10]. In the study by Watanabe et al., the application of TALENs in G. bimaculatus has been described [11]. Although TALENs are simpler in design and have higher specificity, they have certain cytotoxicity and the module assembly process is cumbersome. In previous studies, the mutation rate of knocking out the lac2 gene in G. bimaculatus was 92% by CRISPR/Cas9 mRNA gene editing system [5]. Stable mutant lines were also generated by injection of Cas9 protein in studies of gene function in G. bimaculatus wing development [12].
The cuticle hardening process is very complex and insect cuticle is soft and pale when it is initial synthesized [13]. Tyrosine hydroxylase (TH) is the initial rate-limiting synthetase involved in the biosynthesis of 3,4-dihydroxyphenylalanine (DOPA) during the sclerotization process [14,15]. DOPA is a quinone melanin precursor essential for exoskeletal pigmentation and cuticle sclerosis [16,17]. Ddc is a pyridoxal-5-phosphate-dependent enzyme that catalyzes the conversion of DOPA to dopamine. Dopamine is an important neuro-transmitter [18,19]. Insect major royal jelly proteins (MRJP) or Yellow proteins contain an approximately 300 amino acid-long MRJP domain. They were initially identified in the royal jelly proteins that play a central role in honeybee development [20].
In this article, we target the TH gene and yellow-y gene in the melanin synthesis pathway. A high proportion of TH and yellow-y mutants was obtained, including insertion and deletion mutation types. We randomly selected some yellow-y individuals and wild type (WT) for mating and generation inheritance. We identified mutations and found they were inherited from parents, indicating that the mutations are stable and inheritable. This greatly promotes the operability of the G. bimaculatus as a model system in regulating melanin synthesis, and provides a characteristic animal model for studying the critical roles and protective mechanism of cuticular color.

Insects culturing and egg collection
G. bimaculatus was cultured at 25˚C-30˚C, 12 h light:12 h dark photoperiod, and 50% relative humidity (RH). They were feed comprehensive rabbit food, pumpkin and apples. The male and female G. bimaculatus were reared to adults in individual. Three days before microinjection, three female adults and five male adults were placed in the same 1000 ml plastic box for mating. Three days after mating, spawning device was prepared. Paper towels moistened with tap water with a height of about 1.5-2.5 cm were placed in a 1000 mL plastic box with a height of 8.5 cm. The humidity of the paper towel was 60%. Each box contained three mated female adults. After 1 hour, the crickets were moved to a new box to continue laying eggs. According to statistics, a cricket could lay about 240 eggs at a time.

Identification of G. bimaculatus TH and yellow-y
The amino acid sequence tyrosine hydroxylase is analyzed in combination with some of the TH (BAM15632) genes already reported on NCBI and the transcriptome database of G. bimaculatus [21]. The target site of TH gene is designed in the seventh exon by sequence alignment (Fig 1). The yellow-y gene is searched from the insect-genome database (insect-genome.com), the sequence number is GBI_10058-RA, and its target site is located in the first exon (Fig 1). The TH protein sequence is aligned with the homologous sequences of other twenty-three species and the yellow-y protein sequence is compared with the homologous sequences of other species (S1 and S2 Tables). The phylogenetic tree was constructed using neighbor-joining method and the confidence values of the branch topology were measured from 1000 bootstrapping repeats (Fig 2).

Construction and synthesis of sgRNA
We used the ZiFit (http://zifit.partners.org/zifit/CSquare9Nuclease.aspx) online tool to design sgRNA. To generate sgRNA templates, one unique oligonucleotide (TH-sgF / yellow-y-sgF) contained a T7 polymerase binding site and target sequence. The sgRNA-R contains a partial sgRNA sequence that overlaps sequences and was annealed to a common oligonucleotide. gRNA scaffold was cloned into pMD 19-T vector. Double strand DNA for specific gRNA synthesis was PCR amplified. The reaction mix was composed of: 0.  DNA. In vitro transcription was carried out for 2 h at 37˚C. 1 μL Turbo DNase was added to degrade the DNA template and incubated at 37˚C for 15 min. sgRNAs were then purified by ethanol precipitation with 3M sodium acetate. The quality of sgRNA was checked with agarose gel electrophoresis, and the concentration was measured with the ultra-micro spectrophotometer (NanoDrop One). sgRNAs were diluted to the concentration of 500 ng/μl. They were aliquoted and stored at −80˚C until further use. All primers are listed in Table 1.

In Vitro verification of sgRNA efficiency
Cas9 protein (E365-01A) was purchased from Novoprotein Company. The final injection concentration was 300 ng/μL. The in vitro verification system was composed of: 14 μL of Target gene PCR product, 3 μL of sgRNA, 1 μL of NLS-Cas9 Nuclease and 2 μL of 10 X Reaction Buffer. In the control group, sgRNA was replaced with sterilized water. The reaction conditions were as follows: 37˚C for 30 min, 70˚C for 10 min. Then the cleavage efficiency of sgRNA was detected by 2% agarose gel electrophoresis.

Microinjection
The eggs of 1 h post spawning were neatly placed in the agar plate model. The needle made by the Micropipette Puller (P-97 Sutter, USA) was cut out of a bevel, sharp like a syringe. The sgRNA and Cas9 protein were mixed according to the system: 3.6 μL of sgRNA, 1.8 μL of Cas9 protein, and 0.6 μL of 10X buffer, then added to the needle, and microinjected with a microinjector (PLI 100 A, Harvard, USA). A mixture of Cas9 protein (300 ng/μL) and TH sgRNA (80 ng/μL, 150 ng/μL, 300 ng/μL, 500 ng/μL) was injected into the eggs. The concentration of yellow-y sgRNA was: 300 ng/μL and 500 ng/μL. Injected eggs were incubated at 28˚C, 60% humidity and 12:12 h photoperiod and sprayed with sterile water containing 50 U/ml penicillin and 50 μg/ml streptomycin (15070-063, Thermo Fisher) every day.

Observing and photograph
As previously described, the body color of hatchlings become black after 1 day [4]. The pigmentation process of representative individuals in TH, yellow-y and WT were photographed just after hatching, at 0.5 h, 1 h, 1.5 h, 3.5 h, 24 h and 5 days after hatching, respectively. Five days after hatch, TH mutant, yellow-y mutant and WT were photographed by microscope (Leica M125 stereo microscope) after crickets placed on ice for 5 minutes. In addition, pigmented and representative individuals were photographed their heads for comparison. Mutant and WT adults were photographed by camera (Canon PowerShot G7 X Mark III).

Genomic DNA extraction and identification of mutagenesis
Genomic DNA was extracted from hatched larvae or antennae of adult of the injected eggs with a DNA extraction buffer, incubated with proteinase K, and purified with the AxyPrep ™ Multisource Genomic DNA Miniprep Kit. The PCR conditions were 98˚C for 2 min, followed by 35 cycles of 94˚C for 10 s, 56˚C for 30 s, and 72˚C for 1 min, followed by a final extension period of 72˚C for 10 min. The PCR products were directly sequenced. All primers are listed in Table 1.
Thirteen F 0 nymphs injected with TH gene, 6 F 0 individuals injected with yellow-y gene and four F 1 individuals of yellow-y were randomly selected. These individuals showed double peaks in DNA sequencing around the target sites. PCR products of these individuals were TA cloned to identify the specific type of mutation.

Germline inheritance
Female and male F 0 individuals with either TH or yellow-y mutations were selected and mate with the WT siblings. Three days after mating, females were placed in the spawning device to lay eggs. Eight days after spawning, each group has 100 of the F 1 eggs were randomly selected for DNA sequencing. The rest of the eggs were cultured to adults for strain construction.

In vivo melanization assays
Three 1st-instar nymphs of the same size and significantly lighter cuticles in the TH group mutants were ground at 4˚C with 180 μL PBS (pH = 7.0) and centrifuged (500× g for 5 min at −4˚C). The nymphs of WT group were used as the control. A total of 80 μL supernatant (the Bradford method quantitative protein concentration was 6 mg /mL) was transferred into PCR tube. The A490 value was determined to assess the melanin content.

Dopamine contents and genes expression in cuticle tanning pathway
The dopamine content of TH and WT nymphs at the 1st-instar was determined using a commercial kit (Nanjing Jiancheng Institute of Bioengineering, China). Absorbance of 490 nm was detected in Microplate Spectrophotometer. The samples were homogenized in 1 X PBS and centrifuged at 4˚C and 12000 g for 10 min. Supernatants were used to measure the dopamine concentration according to the manufacturer's protocols. The expression level of Ddc gene involved in the cuticle tanning pathway was detected. The qPCR primer sets are presented in Table 1. Data were normalized for β-actin, and two independent samples were analyzed using Student's t test.

Identification and analysis of TH gene and yellow-y gene
Genomic structure analysis reveals that the TH gene contains twelve exons and eleven introns (Fig 1). The yellow-y gene contains four exons and three introns (Fig 1). Twenty-three orthologous protein sequences reported in other insects and TH of G. bimaculatus are used to construct a phylogenetic tree (S1 Fig). Phylogenetic analysis of the yellow gene of G. bimaculatus shows that it is closely related to the yellow-y gene of other species (S1 Fig).

The effectiveness of TH gene and yellow-y gene targeting
The in vitro verification (Fig 2; S2 and S4 Figs) shows that TH sgRNA and yellow-y sgRNA are effective. The target strip can be cut into two segments of different sizes. The original length of TH gene fragment is 545 bp, which is digested into 464 bp. The original length of yellow-y gene fragment is 1058 bp and is digested into 826 bp. Using Cas9 protein without sgRNA as negative control, non-specific cleavage is excluded.
The results of TH gene microinjection (S3 Table) show that the average hatching rate of 80 ng/μL was 13.00%, and the average mutation rate was 69.23%; the average hatching rate of 150 ng/μL was 10.00%, and the average mutation rate was 90.00%; the average hatching rate of 300 ng/μL was 8.00%, and the average mutation rate was 87.50%; the average hatching rate of 500 ng/μL was 11.00%, and the average mutation rate was 90.91%. The DNA sequencing results also indicate that comparing with WT, the mutants show double peaks or random peaks at the target site, indicating the occurrence of mutations (Fig 2). These experimental results fully prove the effectiveness of the target.
In order to determine the specific targeted mutagenesis of the TH gene by the CRISPR/ Cas9 gene editing system, 13 individuals were randomly selected for TA cloning with DNA sequencing results showing double peaks at target site. The sequencing results show that there are two types of mutations, either deletion or insertion. The specific mutation types are deletion of 6 bp, 21 bp, 4 bp, 5 bp, 1 bp, 3 bp, 5 bp, 4 bp, 2 bp and insertion of 8 bp, 5 bp, 2 bp, 9 bp (Fig 3).
The results of TA cloning of yellow-y F 0 generation show that the types of mutations including deletion of 3 bp and 4 bp (Fig 3). A female and a male with mutations were randomly selected to cross WT and their offspring were identified. Among the offspring of yellow-y ♀ / WT ♂, 90 eggs are sequenced, of which 50 are mutants and 40 are WT. Among the offspring of WT ♀ / yellow-y ♂, 94 eggs are sequenced, of which 50 are mutant and 44 are WT ( Table 2). These results indicate that the CRISPR/Cas9-mediated genome editing system has reliable functions in crickets and can be genetically inherited. We compare the expression levels of TH and yellow-y in WT and F 0 crispants. TH and yellow-y transcripts are significantly down-regulated in mutants (Fig 4).

Phenotypic analysis
TH gene and yellow-y gene are involved in the synthesis pathway of insect melanin [22,23]. Knocking out the TH gene in embryos, the body color of the G. bimaculatus nymphs (5 days after incubation) is mosaic-like in varying degrees (Fig 5; S3 Fig). The individuals of missing three base pairs or multiples that show no obvious changes in body color (35.85%), while the individuals with deletion or insertion neither three base pairs or multiples show more obvious changes in body color (64.15%). Most of these hatched nymphs died before or during the first molting. There is only one individual survives to adult. The legs and wings of the adult are severely defective, which interfere with mating ( Fig 6). Therefore, there is no stable genetic line yet. We tried to cross with WT to obtain F 1 heterozygotes, and the incubation rate of F 1 was significantly reduced. The heterozygotes of F 1 generation successfully incubated were normal in phenotype. But all the F 2 homozygotes died from molting. Therefore, there is no stable genetic line yet.  When the yellow-y gene is knocked out, 71.43% of G. bimaculatus are light brown, with a slight mosaic on the abdomen (S3 Table, Fig 5). This phenomenon could be inherited between germ lines. In the offspring of individuals, the body color of G. bimaculatus has two types: brown or black (Fig 7). Mutants accounted for 53.19% in yellow-y♂ × WT♀ group and 55.60% in WT♂ × yellow-y♀ group ( Table 2).
The pigmentation is partially completed after 24 hours after abolishing of the TH and yellowy genes, while the pigmentation of WT is completed in 1.5 hours (Fig 8). The heads of TH and yellow-y genes mutants are mosaic-shaped, with the TH mutant having a light brown and black mosaic heads, and the yellow-y mutant having a dark brown and black mosaic head (Fig 9).

Suppression of TH impairs G. bimaculatus pigmentation and dopamine synthesis
The in vivo melanization experiments show that the melanin productions of the 1st-instar nymphs of TH mutants are significantly less than that of controls. The values of the absorbance at 490 nm in the control group are: 0.28, 0.192, and 0.24, and those in the experimental group are: 0.125, 0.125, and 0.142 (Fig 10). In agreement with the blockage of cuticle tanning, the dopamine concentration is significantly decreased (Fig 11A) and the expression level of the Ddc gene is also significantly down-regulated in individuals with TH knockout (Fig 11B).

Discussion
In the experiments of the CRISPR/Cas9 gene editing system in Pyrrhocoris apterus, Kotwica-Rolinska et al. pointed out that Cas9 protein as the preferred choice of this approach [24]. The combination of high concentrations of Cas9 and sgRNA has been reported to result in a higher mutation rate but a lower survival rate [25,26]. And injection of the sgRNA and Cas9 protein complex into the zygote forming region enabled Cas9 nuclease to edit the gene immediately after injection [27]. Both of Cas9 plasmid and mRNA could not function immediately [28], they need essential time to experience a protein synthesis process in the embryo [28] and may lower the editing efficiency. In our experiments, the mutation rate is similar to that of previous Cas9 protein systems [12] and mRNA [29]. In addition, the efficiency of mutation is stable. The setting of a series of concentration gradients for the TH gene target is beneficial to the preliminary study and evaluation of the concentration-dependent effects of Cas9 protein and sgRNA on the mutagenesis efficiency and survival rate of G. bimaculatus. However, the study of off-target effects still needs to be carried out and perfected in follow-up experiments. While there are some CRISPR design tools that can predict off-target effects in some genes [30], we cannot rule out the possibility that there are some off-target effects in unassembled duplicate regions. The cuticle is the outermost layer of an insect's body, and its physical properties, such as elasticity, thickness, strength, and stiffness, are key determinants of maintaining movement, mechanical support, body shape, and normal development [31]. As the insect exoskeleton, the cuticle is also the first protective barrier against various environmental stresses and against the penetration of external compounds such as pathogens, dehydration, physical damage and pesticides [32]. However, the rigid insect exoskeleton prevents further growth of molting and metamorphosis. Therefore, cuticle digestion and biosynthesis in insects must occur periodically. Newly synthesized insect cuticle is known to be pale and soft, and must undergo the tanning process (hardening and pigmentation) within a specific period of time for the insect to growth and development [33]. Melanin and quinone pigments derived from catecholamines (such as dopamine) produced by tyrosine-mediated metabolism of cuticle tanning (pigmentation and sclerotization) play a key role in the darkening and hardening of many insect cuticles [23]. TH gene is the initial limiting enzyme for dopamine synthesis. The role of TH and TH homologues in the melanic pigments of holometabolous and hemimetabolous insects is conservative. In Agrotis ipsilon, most CRISPR/Cas9-mediated AiTh knockout embryos can develop but cannot hatch. Successfully hatched embryos died within the first day [34]. Injection of TH dsRNA or feeding TH inhibitors into Tribolium castaneum, Anopheles sinensis and several fruit flies will severely hinder the larval-pupa deformation and adult emergence, and cause the death of most flies [35][36][37]. Although the sequence of the TH gene has been obtained in the study of the serotonin composition of the G. bimaculatus [38], decreased TH gene expression levels by RNAi caused wings to become white [39]. Based on gene alignment, we designed a target in the seventh exon of the TH gene of G. bimaculatus [37]. Our results show that after knocking out the TH gene, the body color showed a significant mosaic and most of them died before or during the first molting. Although only one cricket survived as adult, it    bodies are also defective, such as incomplete molting and defective wings and legs. We tried to cross with WT to obtain F 1 heterozygotes, and the incubation rate of F 1 was significantly reduced. The heterozygotes of F 1 generation successfully incubated were normal in phenotype. But all the F 2 homozygotes died from molting. Therefore, the TH gene is essential for the growth and development of G. bimaculatus. The yellow-y gene is functionally located downstream of the TH gene [40]. In Drosophila, the yellow-y gene is highly expressed in the adult epidermis that produces melanin [22,41,42]. In G. bimaculatus, the body color is brown after the yellow-y gene knocking out. Germline genetic experiments fully demonstrate that the genes knocked out by CRISPR/Cas9 editing system can be inherited. In F 1 generation of yellow-y gene, the number ratio of WT to mutant was close to 1:1 (P = 0.026), so it can be preliminarily inferred that yellow-y gene was located on the autochromosome. However, it is necessary to obtain homozygotes to verify this problem and further verify the function of related genes.
Feeding with TH dsRNA decreased the transcription level of target genes and feeding level of larvae, leading to larvae death Plutella xylostella [43]. In addition, in the study of TH gene function mediated by CRISPR/Cas9 gene editing technology in Agrotis ipsilon, the expression level of TH gene in the mutants was significantly decreased. The CRISPR/Cas9 system works at the genomic level, disrupting gene function more than RNAi inhibits gene expression at the transcriptional level. TH genes show different spatio-temporal expression patterns in different species, which may be closely related to the upstream and downstream regulatory elements [35,44]. TH is involved in the first step of DOPA production from tyrosine [35,45]. In Tenebrio molitor and G. bimaculatus, the Ddc gene is located downstream of the TH gene [39,46]. Similar body color phenotype was obtained from RNAi silencing TH gene and Ddc gene expression levels in G. bimaculatus. In this study we found that the expression level of Ddc gene was significantly down-regulated in TH mutants of 1st-instar nymphs. The directly interaction of the two genes in G. bimaculatus still need further verification. In the follow-up experiment, we will detect the expression levels of TH and Ddc genes at different instars.
In this study, we use CRISPR/Cas9 system to knock out TH gene and yellow-y gene in G. bimaculatus. Significant phenotypes are observed in F 0 mosaic mutants. A phenotype similar to F 0 crispants is observed in the F 1 heterozygotes of the yellow-y gene with some variation. Because TH gene homozygous is lethal, it not only affects the pigmentation of G. bimaculatus, but also may affect the overall development and growth of G. bimaculatus. By now, this complex process and the regulatory signaling pathway needs further elucidate. In subsequent experiments, we will evaluate the causes of TH gene damage to development, focusing on the gene network and molecular interaction of TH genes and the earlier stage. We will also conduct further research on gene functions in the melanin synthesis pathway.
Supporting information S1